Endogenous and ectopic expression of <i>Mgn</i>.

<p><b>(A)</b> Relative expression levels of <i>Mgn</i> and <i>Mash1</i> in mutant to wild-type mice. The bar height and error bars show the mean and standard deviation, respectively. <b>(B)</b><i>Mgn</i> mRNA was expressed at high levels in the vlMB of E12.5 mouse embryos compared with the low level of expression in the dMB determined by ISH, LacZ staining of <i>Mgn</i>^<i>tLacZ</i> mice, and IHC of wild-type embryos with the anti-MGN antibody on coronal sections. The lines indicated the borders of m3-m5 domains. MASH1 protein is also expressed at higher levels in the vlMB compared with its dorsal expression, as determined by IHC with anti-MASH1 antibody. <b>(C)</b> WISH was performed with <i>Gad67</i> probe in ONTC of wild-type E11.5 embryos with <i>Mgn</i> cDNA electroporated in one of the sides (right side), whereas the other side served as a control (left side). The neural tube is opened along the ventral anterior-posterior axis. Lightning bolt icons point to the ectopic expression of <i>Gad67</i> after <i>Mgn</i> electroporation. Subdomains are shown according to the prosomeric model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127681#pone.0127681.ref021" target="_blank">21</a>]. (<b>D</b>) WISH was performed with a <i>Gad67</i> probe in the ONTCs of E11.5 wild-type mouse embryos after 10 h of incubation. The red dashed line delineates the alar/basal (a/b) boundary. The black dashed lines delineate the transversal neural tube domain boundaries. (<b>E and F</b>) E11.5 <i>Mash1</i>^−/− ONTCs 24 h after micro-electroporation in the mesencephalic alar plate with the Mgn-IRES-EGFP plasmid. <b>(E)</b> A fluorescence signal showing the electroporated location. <b>(F)</b> Induction of <i>Gad67</i> expression after electroporation of <i>Mgn</i> cDNA in <i>Mash1</i>^−/− background embryos. (<b>F1</b>) A merged profile of the fluorescence signal and <i>Gad67</i> induction. Scale bars: 500 μm. Abbreviations: a/b, alar/basal plate boundary; d/m, diencephalic–mesencephalic boundary; Hy, hypothalamus; Is, isthmus; p1–p3, prosomeres 1 to 3 in diencephalon; PcP, precommissural domain in the pretectum (p1); rp, roof plate; Zli, zona limitans intrathalamica.</p

MGN formed homodimers and heterodimers with MASH1 in yeast.

<p><b>(A)</b> HEK293 cells were co-transfected, immunoprecipitated (IP), and analyzed by western blotting (WB) with plasmids and antibodies indicated in the IP and WB lines, respectively. The amounts of MGN, MASH1, and NGN2 were verified in total cell lysates (—-) as a control test. Anti-HA antibody (α-HA) was used as a negative control. <b>(B)</b> Y2H analysis of the interactions between MGN and different proneural proteins. AH109 yeasts were co-transformed with a bait and prey plasmid (bait/prey) before being plated on synthetic dropout medium (SD/Leu⁻Trp⁻). (<b>C</b>) The same yeast colonies were plated in the same order on SD/Ade⁻His⁻Leu⁻Trp⁻ (to select positive protein–protein interactions) and used for the ß-galactosidase activity assay (<b>D</b>). Binding domain (BD) alone and activator domain (AD) alone plasmids; BD–human Lamin C provided a control for the AD–prey interaction. P53/SV40 interaction was used as positive control (P53/SV40 was diluted to 1 in 5 in <b>D</b>).</p

Depletion of dMB and vlMB GABAn in the <i>Mgn</i>^{<i>−/−</i>}<i>Mash</i>^{<i>−/−</i>} mice occurred through GABAergic identity failure.

<p><b>(A)</b> Immunohistochemical staining of BrdU on coronal sections showing the distribution of BrdU⁺ cells in the developing dMB and vMB of wild-type (WT) and double mutant mice (<i>−−/−−</i>). <b>(B)</b> Neuronal cell density and cytoarchitectural analysis on the coronal sections of E12.5 embryos using NeuN and the Ca²⁺-binding proteins Calretinin and Calbindin, respectively. There were no significant difference between mean WT and double mutant (NeuN: dorsal WT = 64.22±2.08; dorsal <i>−−/−−</i> = 61.33±2.69; n = 18; p = 0.40. Medial WT = 52.28±3.60; medial <i>−−/−−</i> = 56.72±1.97; n = 18; p = 0.29. Ventrolateral WT = 46.44±2.36; ventrolateral <i>−−/−−</i> = 43.67±2.52; n = 18; p = 0.43); (Calbindin: dorsal WT = 43.28±6.20; dorsal <i>−−/−−</i> = 37.50±5.54; n = 18; p = 0.49. Medial WT = 52.22±5.32; medial <i>−−/−−</i> = 46.56±2.69; n = 18; p = 0.35. Ventrolateral WT = 47.39±3.19; ventrolateral <i>−−/−−</i> = 46.17±3.92; n = 18; p = 0.81). <b>(C)</b><i>Lac</i>Z staining of coronal sections from double mutants and controls at E12.5; C2 and C4 show prolonged staining times. Owing to the long half-life of lacZ protein, signal can also be seen in the intermediate and mantle zones. <b>(D)</b> Immunohistochemical staining with anti-LacZ (in green) and anti-BRN3a (<i>Pou4f1</i>_MGI) (in red, as a morphological marker) antibodies that shows radial migration of GABA-defective neurons through the intermediate zone (IZ) toward the mantle zone (MZ) in double mutants. Scale bars: 100 μm in A, and 200 μm in B-C.</p

<i>Mgn</i> and <i>Mash1</i> were necessary and sufficient to induce GABAn in the MB.

<p>Phenotypic analysis of <i>Mgn</i>^{<i>−/−</i>}<i>Mash1</i>^{<i>−/−</i>} (<i>−−/−−</i>) mice by ISH with the <i>Gad67</i> riboprobe on sagittal <b>(A)</b> and coronal <b>(B-C)</b> sections, showing the mesencephalon at E12.5 and E14.5 from both wild-types (A1–A4; B1 and B2) and double mutants (with B5–B7 being representative slides from anterior to posterior). The arrows in A14, B4, and C4 indicate the presence of few <i>Gad67</i> cells in the m5 domain (m5) at E12.5 and E14.5. Scale bars: 500 μm. Abbreviations: HB, hindbrain; Is; isthmus; SC, superior colliculus; SNpr, substantia nigra pars reticulata; vMB, ventral midbrain; VTA, ventral tegmental area.</p

Colocalization and co-immunoprecipitation of MGN and MASH1 in mice.

<p>Immunohistochemical staining of coronal sections from E12.5 wild-type mouse embryos. <b>(A)</b> Representative confocal photomicrographs of immunohistochemical staining with anti-MASH1 (A2) and anti-MGN (A3) from the dMB (squared area). Nuclei stained with DAPI are shown in blue (A1). A merged picture of A2 and A3 indicating the colocalization of MASH1 and MGN is shown in A4. The MGN⁺ subpopulation represented approximately 50% of the total MASH1⁺ cells of the VZ at this stage. <b>(B-C)</b> MASH1 is an interaction partner of MGN in physiological conditions. Endogenous MGN was IP from the dMB and vMB of E12.5 wild-type mouse embryos with anti-MASH1 antibody and detected by western blotting with anti-MGN antibody (<b>B</b>) and vice versa (<b>C</b>). Lysates from 129SV embryonic stem cells (ES) electroporated with the indicated plasmids, the embryo body (where <i>Mgn</i> is not expressed) and the MB from <i>Mgn</i>^{<i>−/−</i>} and <i>Mash1</i>^{<i>−/−</i>} mutants were used as controls. Equal input and quality of the protein lysate prior to immunoprecipitation was shown by immunostaining with anti-ß-ACTIN antibody. Scale bars: 100 μm.</p

Summary model of the dorsal/ventral mechanisms underlying MB GABAergic neurogenesis.

<p><b>(A)</b> Two genes, <i>Mgn</i> and <i>Mash1</i>, functioned as selectors of GABAergic identity in the MB through different dorsal/ventral mechanistic actions at different times. The MGN/MASH1 heterodimers (green shape coupled with yellow) appear essential for the induction of dorsal GABAn (m1 and m2 domains) that generate GABAn in the colliculi, laterodorsal periaqueductal gray area (ldPAG) and the ventrolateral periaqueductal gray area (vPAG). MGN or MASH1 homodimers (shapes coupled with the same color) are not sufficient to trigger GABAergic identity, and synergistic cooperation between MGN and MASH1 factors to form heterodimers is essential for the acquisition of dorsal GABAergic identity. The mechanism operating in the vlMB area (m3, m4, and m5 domains), which gives rise to the ventral PGA and reticular formation nuclei (MBRf) GABAn, appeared to be different from that in the dorsal area. In this model, the MGN/MASH1 heterodimer is not a prerequisite for the induction of ventrolateral GABAn, with either homodimer being sufficient alone and following a dose-dependent mechanism. The GABAergic phenotype observed in mutant mice is depicted with a green tick or red cross to denote the presence or absence of GABAn, respectively. <b>(B)</b> MB GABAn arise during three waves of neurogenesis, at both specific developmental times and specific locations. The earliest wave of GABAergic neurogenesis (1^st) starts at E10.5 in the presence of <i>Mgn</i> and/or <i>Mash1</i>, without preference. The second phase (2^nd) takes place two days later at E12.5 in the dMB. The third population (3^rd), corresponding to the GABAn of the SNpc and VTA, appears at E14.5. These different mechanisms define a new model for dorsal and ventrolateral GABAergic neurogenesis. Red lines delimitate the embryonic MB into seven subdomains (m1-m7) along the dorsal/ventral axis, characterized by the expression of specific transcription factors [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127681#pone.0127681.ref010" target="_blank">10</a>] and their corresponding MB plates, established by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127681#pone.0127681.ref022" target="_blank">22</a>]. (<i>−−/++</i>; <i>++/−−</i>) stand for <i>Mgn</i> and <i>Mash1</i> single knock-out embryos, respectively. (<i>−+/−+</i>; <i>−−/−+</i>; <i>−+/−−</i>) stand for compound embryos; (<i>−−/−−</i>) stands for double knock-out embryos and <i>++++/−−</i> stands for overexpression of Mgn cDNA in <i>Mash1</i>^{<i>−/−</i>} genetic background embryos. Abbreviations: f, floor plate; r, roof plate; Dap, dorsal alar plate; Lap, lateral alar plate; VLap, ventrolateral alar plate; Lbp, lateral basal plate; Ibp, intermediate basal plate; Mbp, medial basal plate.”</p